We have demonstrated the synthesis and vectorial transport of a cell wall polysaccharide, chitin, in an in vitro preparation. Highly purified plasma membranes isolated from yeast protoplasts were labeled on the outside surface with ferritin. Endogenous chitin synthetase, shown to be exposed on the inner surface of the membrane, was activated by brief trypsin treatment. Incubation of plasma membranes with the chitin precursor, UDP-n-acetylglucosamine, resulted in biochemically demonstrable chitin synthesis. The incubated membranes were examined by electron microscopy and a specific chitin label was used to demonstrate that the newly synthesized chitin was associated exclusively with the outer membrane surface.